During apoptosis, the cell employs mechanisms to achieve a rapid and tidy destruction of the nucleus and genomic DNA. The enzymes which carry out internucleosomal DNA fragmentation, chromatin condensation, and nuclear disassembly are present in a latent form in healthy cells, so mechanisms of inhibiting them during normal cell life are critical. We are studying these mechanisms using a cell-free system in which cytosol from cultured Jurkat human leukemia cells undergoing anti-Fas-induced apoptosis elicits apoptotic changes in purified normal nuclei. We have found apoptotic DNA fragmentation activity to be an endonuclease generated by caspase action during early apoptosis but inactivated in late apoptosis. It has a critical thiol group, chromatographs as a large protein complex, and penetrates nuclei to reach its substrate. Our results suggest that it may be identical to the caspase-activated DNase (CAD or DNA Fragmentation Factor-40) described during the current year (Enari et al. (1998) Nature, 391:43-50). We found that cytosols from several human cell lines (Jurkat, HeLa, SK-N-MC, and WI-38 lines) and mouse tissues contain protein(s) which inhibit the DNA fragmentation activity of apoptotic cytosol. The relationship between this inhibitory activity and the caspase-sensitive inhibitor of CAD, ICAD or DNA Fragmentation Factor-45 described during the current year (Enari et al., ibid.) was studied by the preparation of recombinant His- tagged human DFF45 protein and the generation of a sensitive and specific antiserum against it. On Western blots of cytosolic protein from human cell lines, the antiserum labeled two bands of 44 and 34 kDa, usually major and minor, respectively. These bands were absent in apoptotic cytosol and in healthy cytosol treated with caspase-3. In healthy cells, DFF45 immunoreactivity was found in the cytosol but not in the nucleus or membrane/ organelle fractions. Immunodepletion of DFF45 from healthy Jurkat and HeLa cytosols removed the inhibitory activity, demonstrating that it is DFF45, presumably an excess fraction which is not complexed with DFF40/CAD. Immunodepletion of DFF45 also completely removed caspase-3-activated DNA fragmentation activity, confirming that this latent DNase is entirely bound to DFF45 in healthy cells. These studies confirm and extend the characterization of a mechanism for nuclear destruction which is suppressed during normal cell life, in which a constitutive endonuclease, DFF40/ CAD, binds to an inhibitor, DFF45/ICAD, present in excess. Caspases activated during apoptosis cleave DFF45/ICAD and thereby deinhibit DFF40/CAD, which then enters the nucleus for its destructive mission.